Reassessment of somatostatin receptor SST4 expression in bronchopulmonary and gastroenteropancreatic neuroendocrine neoplasms using the novel rabbit monoclonal anti-human SST4 antibody 7H49L61

Somatostatin receptors SST1, SST2, and SST5 are overexpressed in neuroendocrine neoplasms (NENs), but little is known about SST4 expression in NENs because of a lack of specific monoclonal antibodies. We recently developed and thoroughly characterised a rabbit monoclonal anti-human SST4 antibody, 7H49L61, and showed that it is well suited for identifying SST4 expression in routine pathology samples. The present study aimed to re-evaluate SST4 expression in a large set of NEN samples using this antibody. For this purpose, we assessed SST4 expression in 722 formalin-fixed, paraffin-embedded NEN samples from 274 patients by immunohistochemistry using the novel antibody 7H49L61. The immunostaining was semiquantitatively evaluated using the 12-point immunoreactivity score (IRS), and the results were correlated with clinicopathological data. SST4 was detected in 39.3% of all NENs, but with a median IRS of 2.0, its expression intensity was negligible overall. In all cases, both cytoplasmic and membraneous staining was observed. SST4 expression was somewhat higher in bronchopulmonary NEN (BP-NEN) than in gastroenteropancreatic NEN (GEP-NEN) but still very low. SST4 expression positively correlated with favourable patient outcomes in BP-NEN but had a positive association with Ki-67 index or tumour grading and a negative interrelationship with overall survival in GEP-NEN. In conclusion, unlike that of other SST subtypes, SST4 expression in both BP-NEN and GEP-NEN is negligible and of no diagnostic or therapeutic relevance.

Immunohistochemistry. From the paraffin blocks, 4-µm sections were prepared and floated onto positively charged slides. Immunostaining was performed by an indirect peroxidase labelling method as described previously 36 . Briefly, sections were deparaffinated, rehydrated by means of a descending ethanol series during which endogenous peroxidases were blocked by incubation of the samples for 45 min in 0.3% H 2 O 2 in methanol, microwaved in 10 mM citric acid (pH 6.0) for 16 min at 600 W and incubated with antibody 7H49L61 (1:500) overnight at 4 °C. The primary antibody was detected using a biotinylated anti-rabbit IgG followed by incubation with peroxidase-conjugated avidin (Vector ABC "Elite" kit; Vector Laboratories, Burlingame, CA, USA). The binding of the primary antibody was visualised using 3-amino-9-ethyl carbazole in acetate buffer (Bio-Genex, San Ramon, CA, USA). Sections were rinsed, counterstained with Mayer's haematoxylin, and mounted in Vectamount™ mounting medium (Vector Laboratories, Burlingame, CA, USA). Sections obtained from normal human cortex, pancreas (exocrine pancreas), and placenta were used as positive controls (Supplemental Fig. 1). For negative controls, 7H49L61 was either omitted, replaced by a rabbit IgG isotype control (ab125938; dilution: 1:100; Abcam, Cambridge, UK) or adsorbed for 2 h at room temperature with 10 µg/ml of the peptide used for immunisations (Supplemental Fig. 1). The staining of SST4 in the tumours was assessed using the semiquantitative immunoreactivity score (IRS) according to Remmele and Stegner (1987) 37 . The percentage of positive tumour cells classified in five categories (no positive cells [0], < 10% positive cells [1], 10-50% positive cells [2], 51-80% positive cells [3], and > 80% positive cells [4]) was multiplied by the staining intensity classified in four categories (no staining [0], mild staining [1], moderate staining [2], and strong staining [3]). Thus, IRS values ranging from 0 to 12 were obtained. Only tumour samples with an IRS ≥ 3 were considered SST4-positive.
Statistical analysis. For statistical analysis, SPSS 27.0.0.0 (IBM, Armonk, NY, USA) was used. Because the data were not normally distributed (according to a Kolmogorov-Smirnov test), Kruskal-Wallis test, Mann-Whitney U test, χ 2 test, Kendall's τ-b test, and Spearman's rank correlation were performed. For survival analysis, the Kaplan-Meier method, with a log-rank test and a Breslow test, was used. For all analyses, p values ≤ 0.05 were considered significant. Figure 1 shows representative images of BP-NEN and GEP-NEN samples stained with the anti-SST4 antibody 7H49L61. Figure 2A shows the number of SST4-positive (IRS ≥ 3) or SST4negative (IRS < 3) BP-NEN and GEP-NEN tumours grouped by tumour entity and localisation of the primary tumour. In Fig. 2B, the respective expression levels are depicted, including those of SST4-negative tumours. In all cases, both cytoplasmic and membraneous staining of the cells was observed (Fig. 1). SST4 expression levels varied markedly between individual patients with the same tumour entity or primary tumour localisation, as indicated by the length of the respective boxes and whiskers in Fig. 2B; in some cases, even strong expression (IRS = 12) was observed. SST4 expression also varied distinctly between different samples from the same patient and within one tumour slide.

SST4 expression patterns.
Across all BP-NEN and GEP-NEN tumours, 39.3% of the cases were SST4 positive (IRS ≥ 3). The median IRS, however, was only 2.0 (mean ± S.E.M.: 2.58 ± 0.15), corresponding to negative expression overall. There were, however, some differences in SST4 expression between BP-NEN and GEP-NEN tumours. Whereas 54.8% of BP-NEN tumours were SST4-positive and the overall median IRS was 3.0 (mean ± S.E.M.: 3.41 ± 0.27), corresponding to very low expression, only 32.2% of GEP-NEN cases were SST4-positive and the overall median IRS was 1.5 (mean ± S.E.M.: 2.22 ± 0.18), corresponding to no expression. The differences in SST4 positivity and SST4 expression levels between BP-NEN and GEP-NEN were significant (χ 2 test: p < 0.001; Mann-Whitney test: p < 0.001). There were no differences in positivity rates and SST4 expression levels among the different BP-NEN entities, but SST4 expression varied significantly among GEP-NEN with different localisations of the primary tumour (χ 2 test: p < 0.001; Kruskal-Wallis test: p < 0.001) ( Fig. 2A and B Correlations with clinical data. Because expression and prognostic impact of the other four somatostatin receptors, especially SST1 and SST2, differ between BP-NEN and GEP-NEN 17,18,23 , we analysed correlations between SST4 expression and clinical data separately for the two tumour types. In BP-NEN, SST4 expression did not vary with sex, age, smoking status, or overall survival of the patients; tumour size; the presence of lymph node or distant metastases at diagnosis; or tumour stage or grade. No differences were observed in SST4 IRS between primary tumours and metastases or between tumours from patients who were still alive at the end of the observation period and those who died from tumour-related causes. There was, however, a significant negative correlation between SST4 expression and the expression of the proliferation marker Ki-67 (Table 1). This finding was corroborated by Kaplan-Meier analyses. When using IRS ≥ 3 (the overall median IRS of BP-NEN) as the cut-off value, patients with higher receptor expression had significantly better outcomes than those with lower or no SST4 expression (log-rank test: p = 0.021; Breslow test: 0.007; Fig. 3A).
Similarly, in GEP-NEN, no impact of sex, age, tumour size, lymph node status, presence of distant metastases, or tumour stage on SST4 expression was detected, and no difference in SST4 expression was observed between primary tumours and metastases or between tumours from patients who were still living at the end of the observation period and those who had died. However, functional tumours had significantly lower IRS values compared to non-functional tumours (mean ± S.E.M.  (Table 2) and a negative interrelationship with overall survival (rsp = − 0.376; p = 0.008) were observed. In the respective Kaplan-Meier analyses, when using the overall median IRS of GEP-NEN of 1.5 as the cut-off value between groups, however, no such interrelationship was detected (log-rank test: p = 0.530; Breslow test: p = 0.245; Fig. 3B).  Table 1). Whereas no correlations between SST4 and the other receptors or markers were noted in BP-NEN (Table 1), positive interrelationships between SST4 and SST1, SST3, SST5, CgA, and PD-L1 were observed in GEP-NEN (Table 2). In addition, and similar to previous observations 23,38 , in BP-NEN significant positive correlations between the IRS of SST1 and that of SST5 or of CgA and negative associations between the expression of SST1 and that of CXCR4, Ki-67 or PD-L1 were observed. Additionally, a positive interrelationship between SST3 and SST5  (Table 1).

Discussion
In our study, 39.3% of tumours were SST4-positive, including 54.8% of BP-NEN and 32.2% of GEP-NEN. This corresponds well with the findings of most previous reports 10,11,[13][14][15][16]18,19,[21][22][23][24][28][29][30][31][32][33][34] , but unlike the earlier immunohistochemical studies, our investigation demonstrated for the first time cytoplasmic as well as membraneous staining of tumour cells. In spite of this, the overall median IRS across tumours was only 2.0, and, thus, below the threshold set for receptor positivity. Therefore, SST4 expression seems to be negligible in NEN overall. There were some differences in SST4 positivity rates and SST4 expression levels between BP-NEN and GEP-NEN and also between GEP-NEN tumours with different sites of origin. Nevertheless, the highest median IRS observed across all NEN entities and sites of origin was noted in AC and amounted to only 3.7, corresponding to low expression.
In the present study, we observed a significant negative correlation between SST4 expression and Ki-67 index and significantly better outcomes in patients with higher receptor expression than in those with lower or no SST4 expression for BP-NEN. This corresponds well to literature data showing that SST4 expression decreases with increasing malignancy from AC to SCLC in BP-NEN 22 . Conversely, in GEP-NEN, we noted positive correlations between SST4 expression and tumour grade as well as Ki-67 index, which also fit well with previous reports that SST4 mRNA expression increases with tumour grade in GEP-NEN 33 . Functional GEP-NEN have been reported to be associated with better patient outcomes [39][40][41][42] , which matches well with our result that functional GEP-NENs exhibited significantly lower SST4 expression levels than non-functional tumours. Overall, there seems to be an opposite relationship between SST4 expression and patient outcomes in BP-NEN versus GEP-NEN tumours. Very recently, similar divergent observations have been made for G protein-coupled oestrogen receptor expression in these tumours 43 .

Conclusion
In contrast to other SSTs, SST4 expression in both BP-NEN and GEP-NEN is negligible and of no diagnostic or therapeutic relevance.    License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.